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Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae
C A Wurm1, I E Suppanz, S Stoldt
1Mitochondrial Structure and Dynamics Group, Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Journal of Microscopy
|November 6, 2010
Summary
This study introduces an efficient electroporation method for FlAsH-labelling in budding yeast, reducing incubation time from overnight to under an hour. This optimized live cell imaging technique enhances protein distribution analysis in Saccharomyces cerevisiae.
Area of Science:
- Cell Biology
- Biochemistry
- Microscopy
Background:
- Live cell imaging is crucial for understanding protein dynamics.
- The biarsenical-tetracysteine (FlAsH) system enables protein labelling via a biarsenical probe and a small protein tag.
- Current FlAsH-labelling protocols in Saccharomyces cerevisiae require lengthy overnight incubations, limiting experimental flexibility.
Purpose of the Study:
- To develop and characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells.
- To reduce the labelling time for FlAsH-labelling in Saccharomyces cerevisiae.
- To assess the impact of the new method on labelling efficiency and cell viability.
Main Methods:
- Utilized standard electroporation for introducing FlAsH-EDT(2) into live budding yeast cells.
- Compared the new method with established overnight incubation protocols.
- Evaluated labelling efficiency and cell viability post-electroporation.
Main Results:
- Electroporation significantly reduced labelling time to less than 1 hour, compared to over 12 hours previously.
- The reduced incubation time did not compromise labelling efficiency.
- Cell viability remained unaffected by the electroporation-based labelling method.
Conclusions:
- Electroporation provides an efficient and rapid method for FlAsH-labelling in live budding yeast.
- This optimized approach expands the utility of FlAsH-labelling for cells in various growth phases and conditions.
- The method holds potential for adaptation to other high-affinity probes, broadening labelling possibilities in yeast.
